The overall goal of this project is to investigate strategies for the drug treatment of epilepsy through pharmacological studies in animal models and clinical investigation in human subjects. Research was continued evaluating the role of neuroactive steroids in epilepsy and their possible uses in epilepsy therapy. Neuroactive steroids are endogenous steroid hormones (and their synthetic analogs) that rapidly alter the excitability of neurons by direct actions on membrane ion channels, including GABA-A and NMDA receptors. In prior reporting periods, we confirmed that the reproductive hormone progesterone has powerful anticonvulsant activity and we demonstrated that this results from its conversion to the neuroactive steroid allopregnanolone. We proposed that perimenstrual catamenial epilepsy, the increase in seizure frequency that many women with epilepsy experience near the time of menstruation (when progesterone levels fall) may, in part, be related to withdrawal of allopregnanolone. At present, there is no specific treatment for catamenial epilepsy. However, our studies with an animal model of catamenial epilepsy suggested that neurosteroid replacement could be useful. We have initiated clinical studies to validate the neurosteroid withdrawal hypothesis of catamenial epilepsy and we plan a clinical trial to evaluate the utility of neurosteroid replacement. In addition, we have investigated the role of neurosteroids in stress-induced alterations in seizure susceptibility, focusing specifically on tetrahydrodeoxycorticosterone (DOC), an adrenal steroid whose synthesis is enhanced during stress. Our results demonstrated that DOC is a mediator of the physiological effects of acute stress that could contribute to stress-induced changes in seizure susceptibility through its conversion to neurosteroids with modulatory actions on GABA-A receptors including THDOC and possibly also dihydrodeoxycorticosterone (DHDOC). Our results further suggest a role for neuroactive steroids as a treatment approach for stress-related seizures. In the present reporting period we developed an in vitro model to assess neurosteroid effects on seizure susceptibility in the absence of confounding factors such as differences in absorption, metabolism, and brain accessibility. The neurosteroids allopregnanolone and its 5beta-epimer pregnanolone, and pregnenolone sulfate (PS) were examined for effects on spontaneous epileptiform discharges induced by picrotoxin (PTX) and 4-aminopyridine (4-AP) in the CA3 region of the rat hippocampal slice. At a low concentration, allopregnanolone partially reduced PTX-induced bursting and at higher concentrations completely suppressed bursting. In contrast, pregnanolone failed to alter the discharge frequency. Allopregnanolone depressed 4-AP-induced bursting with similar potency as in the PTX model; pregnanolone was also partially effective. In the 4-AP model, allopregnanolone inhibited both the more frequent predominantly positive-going potentials as well as the less frequent negative-going potentials that may be generated by synchronous GABAergic interneuron firing. PS enhanced the PTX bursting frequency and, in the 4-AP model, increased the frequency of negative potentials but did not alter the frequency of positive potentials. By itself, PS did not induce bursting. The effects of the steroids in the in vitro seizure models largely correspond with their activities on GABA-A receptors; suppression of discharges may occur as a result of direct activation of these receptors rather than modulation of GABA-mediated synaptic responses. We conclude that PTX and 4-AP-induced bursting in the hippocampal slice are useful models for directly assessing neurosteroid effects on seizure susceptibility under conditions that eliminate the factor of brain bioavailability.